The Key Factors of Immunoprecipitation

It is known that immunoprecipitation is a method to test target protein or antigen in solution. This method has been used in the research of protein, gene and other biological areas. It has a important place in biological experiments. Every method to immunoprecipitation has its certain operation process, but most of them are based on the specificity between antibody and antigen. The normal way to use the covalently bound Protein A , the agarose or magnetic microspheres of protein G to purify the compound of antigens and antibody. Thus protein A and protein G can bind with the conserved region of antibodies to form a steady compound of antigens and antibodies which is attached to magnetic microspheres. When the unrelated substance in solution is removed by washing magnetic microspheres, antigens or molecules bound with antigens can be purified. When do this experiment, some important factors need to be taken into account.
First factor is specimen. The key of the success of immunoprecipitation is primary sample preparation. Immunoprecipitation experiment aims to test the interaction between antibody and antigen, so the quality of sample determines the quality of the interaction between antigens and antibodies. Preparing samples needs to choose an appropriate way according to the resource of antibody samples including tissue samples and cells. Then place the antigens which is to be tested in sample solution to release. It requires adding proper amount of protease into lysis buffer to avoid degradation of antigens and compounds. In addition, it is suggested to choose the lysis buffer containing proper disincrustant to make sure that these cells can release antigens.
The second significant factor is antibody. When choose antibody, not only the specificity but also affinity of antibodies have to be taken into account. Polyclonal antibody only has a good specificity to single epitope of antigen and it may cause that epitope of antigen can not get enough exposure and get damaged. Thus antibody cannot recognize antigen, which stocks producing immune complex. It will have a bad effect in the result of immunoprecipitation. However, monoclonal antibody or mixed polyclonal antibody can bind with several epitopes of antigen. They can solve the problem of affinity. But not all kinds of antibodies can be applied to immunoprecipitation, so pay more attention to
choosing antibody.
Third factor is magnetic microspheres. The microspheres which are used widely in immunoprecipitation is agarose and magnetic microspheres. They all have its unique features. But the latter becomes rising star, because the heart is superparamagnetism grain and the it is wrapped by a smooth substance. It can binds with more micro proteins and separate the target proteins. So magnetic microspheres can save much time and it earns good reputation.

A Brief Introduction of Immunoprecipitation

A Brief Introduction of Immunoprecipitation

What is Immunoprecipitation?

Protein is always the focus of research for global scientists for it is the most important molecule in living forms. Immunoprecipitation is a classical method to researching interaction different proteins on the basis of specificity between antibody and antigen and it is also regarded as an effective way to recognize the mutual physiological action between two kinds of proteins in cells. It presents that the immune complexes produce protein precipitation in some certain condition after the specific binding between soluble antigen and antibody in liquid or gel. Immunoprecipitation, agglutination, neutralization reaction and the reaction between antigen and antibody are called classical immunological techniques. With the deepening of the protein research, more complicated methods are produced on the basis of combination of immunoprecipitation and other methods. Thus methods of protein analysis become more various and its application area become wider. This technology has a wide application in gene, protein, and their reaction.

Development

Basic strategy of Immunoprecipitation is that utilizing the high affinity in antibody and antigen to test and bind the target molecule of liquids. Immunoprecipitation is advancing with the development of science and technology. In early stage, researchers use gel electrophoresis to test immunoprecipitation protein. They add antigen solution into holes of permeable matrix and add antisera into nearby holes. With the spreading of antigens and antibodies, macromolecular protein complexes separate out as the precipitation line which can be recognized by naked eyes in the solution. The early improvement of immunoprecipitation is to promote polymer reaction to make immune complexes separate out from the solution. Later, researchers found that using secondary antibody and immune complexes’ antibody to build a net can save the step of titration experiments of primary antibody of every antigens. This methods is still in use. In 70s in 20th century, people start use solid phase reaction. They protein A fixed in staphylococcus aureus to adsorbent antibody and bind with certain antigen. With the development of science and technology, it evolves. The microspheres of protein A or G are used to separate antibody complex from antigen complex to test antigen or target protein.